MiR-378b Modulates Chlamydia-Induced Upper Genital Tract Pathology

Genital Chlamydia trachomatis infection causes severe reproductive pathologies such as salpingitis and pelvic inflammatory disease that can lead to tubal factor infertility. MicroRNAs (miRNAs) are evolutionarily conserved regulators of mammalian gene expression in development, immunity and pathophysiologic processes during inflammation and infection, including Chlamydia infection. Among the miRNAs involved in regulating host responses and pathologic outcome of Chlamydia infection, we have shown that miR-378b was significantly differentially expressed during primary infection and reinfection. In this study, we tested the hypothesis that miR-378b is involved in the pathological outcome of Chlamydia infection. We developed miR-378b knockout mice (miR-378b−/−) using Crispr/Cas and infected them along with their wild-type (WT) control with Chlamydia to compare the infectivity and reproductive pathologies. The results showed that miR-378b−/− mice were unable to clear the infection compared to WT mice; also, miR-378b−/− mice exhibited a relatively higher Chlamydia burden throughout the duration of infection. However, gross pathology results showed that miR-378b−/− mice had significantly reduced uterine dilatations and pathologic lesions after two infections compared to WT mice. In addition, the pregnancy and fertility rates for infected miR-378b−/− mice showed protection from Chlamydia-induced infertility with fertility rate that was comparable to uninfected WT mice. These results are intriguing as they suggest that miR-378b is important in regulating host immune responses that control Chlamydial replication and drive the inflammation that causes complications such as infertility. The finding has important implications for biomarkers of Chlamydial complications and targets for prevention of disease

Molecular Analysis of Retroviral Transduction in Chronic Myelogenous Leukemia

Overview summary A human gene transfer clinical protocol has been approved by the RAC and the FDA in which purged remission marrow cells of adult patients suffering from chronic myelogenous leukemia are marked with the retroviral vector LNL6 prior to re-infusion into the patient at the time of relapse (see this issue, pp. 359–376). Claxton et al. provide here a portion of the data that was used to obtain approval for this protocol.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63433/1/hum.1991.2.4-317.pd

Role of Epithelial-Mesenchyme Transition in Chlamydia Pathogenesis.

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy

<i>Chlamydia</i> infection of reproductive epithelial cells induces epithelial-mesenchyme transition (EMT) (mesenchymal markers).

<p>Monolayers of murine oviduct epithelial cells were infected with <i>C</i>. <i>trachomatis</i> L and after 72h immunofluorescence staining of the cells for EMT markers was performed on infected and non-infected monolayers by standard procedures. Fluoresceinated antibodies against the following markers were used: T-cadherin, Snail1/2, fibronection, Zeb1 and MMP9. Antibodies stained both intracellular and surface antigens. Images were acquired at 20X objective on a Nikon fluorescent microscope with the same microscope settings and exposure time. The representative slides are from several repeated experiments showing identical results.</p

<i>Chlamydia</i> infection caused the altered expression of proteins that regulate epithelial functional integrity and pathologic EMT in the reproductive system (Down-regulation).

<p>Results were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145198#pone.0145198.g006" target="_blank">Fig 6</a> and selected molecules that were down-regulated have been presented.</p

Chlamydial genital infection caused a sustained alteration of key miRNAs that control the functional integrity of epithelial cells (down-regulated miRNAs).

<p>Analysis of the pattern of expressed miRNAs in the oviducts of infected infertile mice and non-infected mice employed a combination of microarray and quantitative real time PCR as per Signosis’ miRNA Array IV service (Signosis, Inc. Sunnyvale, CA), as described in the Materials and Methods section. The labeled oligonucleotide probe mixture corresponded to 540 randomly selected miRNAs which have been reported to play a role in inflammation, apoptosis and cancer by literature search [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145198#pone.0145198.ref101" target="_blank">101</a>]. Quantitative Real-Time PCR was used to validate the microarray data using miRNA-specific oligo mix. Information about miRNA target mRNA genes was obtained from several Web-accessible miRNA database searching programs, including <a href="http://www.microrna.org" target="_blank">http://www.microrna.org</a>, <a href="http://www.miRBase.org" target="_blank">http://www.miRBase.org</a>, <a href="http://www.targetscan.org" target="_blank">http://www.targetscan.org</a> and published reports [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145198#pone.0145198.ref102" target="_blank">102</a>]. Results presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145198#pone.0145198.t001" target="_blank">Table 1</a><b>(are shown as Fig 1 Down-regulated</b>) were derived from oviducts harvested from 1 wk through at 80 days (11.4 wk) post-infection. Experiments were repeated 4 times with at least 6 oviducts in each group. Statistically significant changes had <i>P value</i> from ≤ 0.05.</p

Caspase inhibition prevented chlamydial-induced EMT.

<p>Monolayers of murine oviduct epithelial cells were infected with <i>C</i>. <i>trachomatis</i> L and after 72h immunofluorescence staining of the cells for epithelial and EMT markers was performed on infected and non-infected monolayers by standard procedures. Z-VAD-FMK and the control Z-FA-FMK were used in culture at 50 micromole. Quantification of fluorescence was performed by scanning fluorescent-stained cells at 20X objective on a Nikon fluorescent microscope using the NIS-Elements Imaging Software version 3.20 (Nikon Instruments Inc, Melville, NY USA). Images were acquired for experimental and control cultures with the same microscope settings, exposure time and background. The mean fluorescence intensity and standard deviations of 6 out of 10 scanned fields per slide were calculated automatically by the software. Plotted data were derived from 5 independent experiments.</p

<i>Chlamydia</i> infection of reproductive epithelial cells induces epithelial-mesenchyme transition (EMT) (Loss of epithelial markers).

<p>Monolayers of murine oviduct epithelial cells were infected with <i>C</i>. <i>trachomatis</i> L and after 72h immunofluorescence staining of the cells for epithelial markers was performed on infected and non-infected monolayers by standard procedures. Fluoresceinated antibodies against the following markers were used: E—cadherin and occlusin. Antibodies stained both intracellular and surface antigens. Images were acquired at 20X objective on a Nikon fluorescent microscope with the same microscope settings and exposure time. The representative slides are from several repeated experiments showing identical results.</p

<i>Chlamydia</i> infection caused the altered expression of proteins that regulate epithelial functional integrity and pathologic EMT in the reproductive system (up-regulation).

<p>The quantitative differential proteomics with isobaric tags for relative quantitation (iTRAQ) labeling and LC-MS analysis was used to identify proteins that were differentially expressed in the oviducts from chlamydial infected mice, as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145198#pone.0145198.ref103" target="_blank">103</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145198#pone.0145198.ref104" target="_blank">104</a>]. Briefly, trypsinized proteins extracts were labeled using the 114, 115, 116 and 117 iTRAQ tags and analyzed by MS, as described in the materials and Methods section. Protein/peptide identification and quantification were performed with the Protein Pilot Software version 4.5 and MASCOT, including the search of the database for murine or human proteins (<a href="http://www.uniprot.org" target="_blank">http://www.uniprot.org</a>). Fold Change represented protein expression in infected oviducts divided by protein expression in uninfected oviducts. The down-regulated molecules were expressed in percentage to avoid negative values. Only protein identified by at least 2 peptides and with a P-value < 0.05 were used for quantitation. The biological and functional properties of the proteins and their physiological and disease pathways were established through <a href="http://www.elsevier.com/online-tools/pathway-studio" target="_blank">http://www.elsevier.com/online-tools/pathway-studio</a> (Pathway studio 10; <a href="http://www.elsevier.com/online-tools/pathway-studio" target="_blank">www.elsevier.com/online-tools/pathway-studio</a>) and other specialized biomedical databases that include PubMed and Medline searches. <b>Results show</b> molecules upregulated at D14 and D28 p.i.</p

Role of T cell-derived TNF-alpha in chlamydial-induced infertility.

<p>Groups of female mice (TNF-alpha^-/-, controls TNF-alpha^+/+, and TNF-alpha^-/- that received 10⁷ purified T cells from TNF-alpha⁺⁺ mice) were infected intravaginally with <i>C</i>. <i>trachomatis</i> L2 (WT-L2), as described in the Materials and Methods section. Infected and noninfected control mice were mated and scored for pregnancy and no. of pups. The plotted data are the means (SEM) for groups from 2 independent experiments with 6 mice per experimental group.</p